Recombinant forms of insulin, insulin analogues and/or derivatives have been produced in various microbial expression systems. Currently organisms such as E. coli, S. cerevisiae have been employed for the commercial production of recombinant human insulin and derivatives thereof. Owing to certain disadvantages of these systems such as low expression levels, difficulties in down stream purification etc., the use of methylotrophic yeast Pichia pastoris has been favored as a protein expression system. The expression system offers several advantages such as high expression, simple processing, low production cost, high density culture (U.S. Pat. No. 6,800,606).
Yeast expression systems are popular because they are easy to grow, are fast and scalable; however, some yeast expression systems have produced inconsistent results, and it is sometimes difficult to achieve high yields. One yeast expression system that has shown great promise is the methylotrophic yeast, Pichia pastoris. Compared to other eukaryotic expression systems, Pichia offers many advantages because it does not have the endotoxin problem associated with bacteria or the viral contamination problem of proteins produced in animal cell culture (Cino, Am Biotech Lab, May 1999).
Albeit various advantages are attributed to yeast based expression systems such as Pichia pastoris, one of the major disadvantages of this system is the post-translational modification of resulting proteins which later exist as impurities in the final product that is difficult to purify. Although there are a number of post translational modifications of proteins known, the most common form of post translational modification is glycosylation. (Hart G. W, Glycosylation, Curr. Opin. Cell. Biol 1992; 4: 1017). Glycosylation can be either N-linked or O-linked depending on the expression system. (Gemmill T R et al., Overview of N- and O-linked oligosaccharide structures found in various yeast species, Biochemica et Biophysica Acta, 1999; 1426:227). Glycosylation affects stability of protein conformation, immunogenicity, clearance rate, protection from proteolysis and improves protein solubility. (Walsh G, Biopharmaceutical benchmarks 2006, Nature Biotechnology, 2006; 24:769).
In yeasts, the modification of the sugar branches in the Golgi apparatus involves a series of additions of mannose residues by different mannosyl transferases (“outer chain” glycosylation). The structure of the outer chain glycosylation is specific to the organisms. Such glycosylations are often undesired since it leads to heterogeneity of a recombinant protein product in both carbohydrate composition and molecular weight, which may complicate the protein purification. It may also lead the protein to be become highly immunogenic or can provoke allergic reactions which are undesirable.
Despite great advances in improving biotechnological manufacturing, no global solutions exist for every protein. The manufacturing process for a specific therapeutic protein requires novel and innovative solutions to problems that may be specific for that protein or family of proteins.
Therefore, it is desirable to genetically engineer methylotrophic yeast strains such as Pichia pastoris in which glycosylation of proteins can be manipulated, essentially reduced and from which recombinant glycoproteins can be produced having a mammalian-like post translation pattern without affecting the productivity of the desired end product.